A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

Suzannah J. Rihn(MRC University of Glasgow Centre for Virus Research), Andres Merits(University of Tartu), Siddharth Bakshi(MRC University of Glasgow Centre for Virus Research), Matthew L. Turnbull(MRC University of Glasgow Centre for Virus Research), Arthur Wickenhagen(MRC University of Glasgow Centre for Virus Research), Akira J. T. Alexander(MRC University of Glasgow Centre for Virus Research), Carla Baillie(University of Dundee), Benjamin Brennan(MRC University of Glasgow Centre for Virus Research), Fiona Brown(University of Dundee), Kirstyn Brunker(University of Glasgow), Steven R. Bryden(MRC University of Glasgow Centre for Virus Research), Kerry Burness(University of Dundee), Stephen Carmichael(MRC University of Glasgow Centre for Virus Research), Sarah Cole(MRC University of Glasgow Centre for Virus Research), Vanessa M. Cowton(MRC University of Glasgow Centre for Virus Research), Paul Davies(University of Dundee), Christopher Davis(MRC University of Glasgow Centre for Virus Research), Giuditta De Lorenzo(MRC University of Glasgow Centre for Virus Research), Claire L. Donald(MRC University of Glasgow Centre for Virus Research), Mark Dorward(University of Dundee), James I. Dunlop(MRC University of Glasgow Centre for Virus Research), Matthew Elliott(University of Dundee), Mazigh Fares(MRC University of Glasgow Centre for Virus Research), Ana da Silva Filipe(MRC University of Glasgow Centre for Virus Research), Joseph R. Freitas(Griffith University), Wilhelm Furnon(MRC University of Glasgow Centre for Virus Research), Rommel J. Gestuveo(University of the Philippines Visayas), Anna Geyer(University of Dundee), Daniel M. Giesel(University of Dundee), Daniel Goldfarb(MRC University of Glasgow Centre for Virus Research), Nicola Goodman(University of Dundee), Rory Gunson, C. James Hastie(University of Dundee), Vanessa Herder(MRC University of Glasgow Centre for Virus Research), Joseph Hughes(MRC University of Glasgow Centre for Virus Research), Clare Johnson(University of Dundee), Natasha Johnson(MRC University of Glasgow Centre for Virus Research), Alain Kohl(MRC University of Glasgow Centre for Virus Research), Karen Kerr(MRC University of Glasgow Centre for Virus Research), Hannah Leech(University of Dundee), Laura Sandra Lello(University of Tartu), Kathy Li(MRC University of Glasgow Centre for Virus Research), Gauthier Lieber(MRC University of Glasgow Centre for Virus Research), Xiang Liu(Griffith University), Rajendra Lingala(Indian Immunologicals (India)), Colin Loney(MRC University of Glasgow Centre for Virus Research), Daniel Mair(MRC University of Glasgow Centre for Virus Research), Marion McElwee(MRC University of Glasgow Centre for Virus Research), Steven McFarlane(MRC University of Glasgow Centre for Virus Research), Jenna Nichols(MRC University of Glasgow Centre for Virus Research), Kyriaki Nomikou(MRC University of Glasgow Centre for Virus Research), Anne Orr(MRC University of Glasgow Centre for Virus Research), Richard Orton(MRC University of Glasgow Centre for Virus Research), Massimo Palmarini(MRC University of Glasgow Centre for Virus Research), Yasmin A. Parr(MRC University of Glasgow Centre for Virus Research), Rute Maria Pinto(MRC University of Glasgow Centre for Virus Research), Samantha Raggett(University of Dundee), Elaine Reid(University of Dundee), David L. Robertson(MRC University of Glasgow Centre for Virus Research), Jamie Royle(MRC University of Glasgow Centre for Virus Research), Natalia Cameron-Ruiz(MRC University of Glasgow Centre for Virus Research), James G. Shepherd(MRC University of Glasgow Centre for Virus Research), Katherine Smollett(MRC University of Glasgow Centre for Virus Research), Douglas G. Stewart(MRC University of Glasgow Centre for Virus Research), Meredith Stewart(MRC University of Glasgow Centre for Virus Research), Elena Sugrue(MRC University of Glasgow Centre for Virus Research), Agnieszka M. Szemiel(MRC University of Glasgow Centre for Virus Research), Aislynn Taggart(MRC University of Glasgow Centre for Virus Research), Emma C. Thomson(MRC University of Glasgow Centre for Virus Research), L. Tong(MRC University of Glasgow Centre for Virus Research), Leah S. Torrie(University of Dundee), Rachel Toth(University of Dundee), Margus Varjak(MRC University of Glasgow Centre for Virus Research), Sainan Wang(University of Tartu), Stuart G. Wilkinson(University of Dundee), Paul G. Wyatt(University of Dundee), Eva Žusinaite(University of Tartu), Dario R. Alessi(University of Dundee), Arvind H. Patel(MRC University of Glasgow Centre for Virus Research), Ali Zaid(Griffith University), Sam J. Wilson(MRC University of Glasgow Centre for Virus Research), Suresh Mahalingam(Griffith University)
PLoS Biology
February 25, 2021
10.1371/journal.pbio.3001091
Cited by 242Open Access
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Abstract

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

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